Rapid molecular methods for the strain-level identification and tracking of drug-resistant bacteria are essential to the diagnosis and treatment of infectious disease and to hospital infection control strategy. Our work aims to develop novel applications of mass spectrometry to these problems in the clinical microbiology laboratory. Our approach involves both the identification and characterization of new bacterial protein markers and development of mass spectrometry-based assays to detect there markers using LC-MS/MS, Q-TOF, QQQ, MALDI-TOF and MRM technologies. Previously published work involved identification of a peptide marker for tracking a carbapenemase-carrying resistance plasmid that could be detected by MALDI-TOF mass spectrometry (Lau et al, 2014), followed by a clinical validation study demonstrating the method's utility in identifying KPC carbapenemase proteins in clinical isolates from NIH Clinical Center patients (Youn et al, 2016). Work done during the current fiscal year in collaboration with Anthony Suffredini's group in CCMD, NIHCC included the development of a genoproteomics approach for identifying strain-specific peptide markers based on LC-MS/MS profiling of digested peptides (Wang et al, 2016). This work provides a general method for bacterial strain typing by mass spectrometry using an in silico computational approach to guide selection of informative, genome-specific tryptic peptides from LC-MS/MS experimental profiles. The method is generalizable to other bacterial species, and the peptides may be useful for rapid mass spectrometry strain tracking of isolates with broad application to infectious disease diagnosis. Work is in progress to apply this approach to the direct detection of bacteria in primary specimen matrices.